Meningococcal meningitis is the disease identified as the inflammation of meningitis of brain and spinal cord. The causing agent behind bacterial meningitis is Neisseria meningitidis. Clinical manifestation of meningococcal infection includes fever, headache, photophobia, neck stiffness, occasional seizures, altered mental status, nausea, vomiting, myalgia, and petechial or purpuric rash (28-77%). Despite antibiotic therapy the mortality rate is higher and sometimes consequences are permanent. After sequel consequences may include loss of hearing, diminished vision or loss of limb due to gangrene formation. Neisseria meningitidis are the gram-negative diplococci which were identified on the basis of their capsular polysaccharides. So far 12 antigenic distinct capsular groups has been identified according to characteristics of the polysaccharide capsule for this microorganism, which include Neisseria meningitidis A, Neisseria meningitidis B, Neisseria meningitidis C, Neisseria meningitidis E, Neisseria meningitidis H, Neisseria meningitidis I, Neisseria meningitidis K, Neisseria meningitidis L, Neisseria meningitidis W135, Neisseria meningitidis X. Neisseria meningitidis Y, and Neisseria meningitidis Z. They can be further distinguished to various types and sub type based on the outer membrane proteins. They are also identified as various sequence type (ST) based on the sequencing of certain regions of their chromosomal DNA. Out of 12, six capsule types (Neisseria meningitidis A, Neisseria meningitidis B, Neisseria meningitidis C, Neisseria meningitidis Y, Neisseria meningitidis W135 and Neisseria meningitidis X) were found responsible for the invasive meningitis and septicemia world wide. The relative importance of each serogroups varies with geographic region. Therefore, determining serogroup responsible for the sporadic case is important for containment of disease in a particular geographical location. The cause of disease development in some carriers is not completely understood. However, few identified risk factors are age, season, smoking, preceding influenza A infection and living in ‘closed’ or ‘semi-closed’ communities (Cartwright, 1995). Meningococcal infection establishes harmless commensal relationship colonizing the nasopharynx among 25% adolescents and 5 to 11% of human adults without any symptoms of illness. But in the case of infection among infants and young children, the carriage rate is low as compared to adults (Christensen et al., 2010). The mode of meningococcal transmission is by aerosol, droplets or direct contact with respiratory secretions of someone carrying the organism. Transmission usually requires either frequent or prolonged close contact like exposure to the bacteria. There is a marked seasonal variation in meningococcal disease, with peak levels in the winter months declining to low levels by late summer.
Meningococcal meningitis is a major public health problem in many countries. As per the WHO estimates world wide annual burden of meningococcal disease is approximately 300,000 to 350,000 cases. The incidence is much higher in many developing countries (about 25/100,000) compared to the US or Western Europe (1-4/100,000). Serogroup A was the cause of most meningococcal disease in the earlier 20th century and is now responsible for recurring epidemics in developing countries, particularly in sub-Saharan Africa. From 2000 to 2002, epidemics of serogroup W-135 occurred, related to spread during the hajj pilgrimage, affecting the health of these travelers and their contacts in countries throughout the world. Serogroup B is the most important cause of endemic disease in developed countries (30-40%) and 80% of disease in the United States and European countries, respectively), and serogroup C has variable rates of endemic occurrence (currently around 30%) in industrialized countries (Jackson et al., 1995). Serogroup Y has emerged in the last decade in the United States and caused one-third of the disease related to meningococcal infections in the country (Rosenstein et al., 1999), usually affecting older age groups. Interestingly, meningococcal pneumonia is usually due to serogroups Y and W-135. Serogroup X has recently been found associated with increase in meningococcal disease outbreak in African meningitidis belt. The outbreaks were reported from Niger, burkina Faso, Tongo and Ghana. In 2010 over 6500 meningitidis cases were reported alone in Burkina Faso out of which 1000 cases were found associated with Serogroup X. Again in 2011 out of 3155 cases of meningitidis, 60% were linked to the Meningitidis X serotype.
Hence, based on the varied epidemiology of the disease a number of commercial vaccines has been made available till date based on the region of occurrence of specific serotypes of Neisseria meningitidis.
TABLE 1Commercial Meningococcal Vaccines available in the marketSr.Name ofType ofSerogroupsCountries whereNo.TradenamemanufacturerVaccineCoveredlicensed 1Bexsero ®GSKRecombinantBUSA, Europe, Australia 2Menactra ®Sanofi PasteurConjugateA, C, Y, W135USA, Canada, India,Australia, Arab andGulf countries 3MenHibrix ®GSKConjugateC, YUSAand Haemophilusinfluenzae type bPRP 4Menitorix ®GSKConjugateCEurope, Australia 5Menomune ®Sanofi PasteurPolysaccharideA, C, W135, YUSA 6Menveo ®NovartisConjugateA, C, W135, YUSA, Canada, Europe,(MCV4 type)and Australia 7Trumenba ®PfizerRecombinantBUSA, Europe,Australia, Canada 8MenAfriVac ®Serum InstituteConjugateAIndia and Africaof India(Lyophilized) 9Mencevax ®PfizerPolysaccharideAApproved in 79(Lyophilized)countries across Africa,Asia, Australia, Europe,Latin America, MiddleEast and New Zealand.10NmVac4-J.N InternationalConjugateA, C, W135, YGulf CountriesA/C/Y/W-Medical135 ®Corporation11Nimenrix ®Pfizer CanadaConjugateA, C, W135, YApproved for sale inmore than 61 countriesincluding the EuropeanEconomic Area,Canada and Australia12VA-Finlay InstituteOuterBCuba, Brazil, ElMENGOC-MembraneSalvador, Nicaragua,BC ®Vesicle Proteinthe DominicanRepublic, Colombia,Argentina and Syria13MeNZB ™ChironOuterA specific strain Only in NewzealandMembraneof serogroup B(Immunization ProgramVesicle Proteinended in 2011 andvaccine is not availablein Newzealandanymore)
Thus we see that, a majority of the vaccine available against Meningococcal meningitis is available only in the developed nations in the Western World, and very few are available in the developing nations, or low or middle economy countries. However, epidemiological studies report that Africa includes at least 25 countries having the highest annual incidence of meningococcal disease in the world. Majority of the diseases are caused by serogroup A meningococcal infection, though it has been reported that serogroups C, W-135 and X are also prevalent. It has been reported that the rate of meningococcal diseases can be as high as 224 incidences/100,000 population in this belt. The South American nations has also shown high rate of prevalence of serogroups B and C (Jafri, R., Ali, A., Messonnier, N., Tevi-Benissan, C., Durrheim, D., Eskola, J., Fermon, F., Klugman, K., Ramsay, M., Sow, S., Zhujun, S., Bhutta, Z. and Abramson, J. (2013). Global epidemiology of invasive meningococcal disease. Popul Health Metrics, 11(1), p. 17.). In the Eastern Mediterranean region, the predominant serogroups are A and W-135. Countries like Sudan and Saudi Arabia in the Eastern Mediterranean are the main sufferers. In most of the Asian countries, few studies have shown that the burden of the diseases in these developing countries may be significant. Apart from the prevalent serogroup A, diseases resulting from serogroups C, Y and W-135 have been reported. There have been meningococcal epidemics in many countries like Cambodia, China, Hong Kong, Indonesia, Malaysia, etc. India has also experienced repeated meningococcal serogroup A epidemics over the last 2 to 3 decades. (VYSE, A., WOLTER, J., CHEN, J., NG, T. and SORIANO-GABARRO, M. (2011). Meningococcal disease in Asia: an under-recognized public health burden. Epidemiol. Infect., 139(07), pp. 967-985.)
Hence, it is established that Meningococcal infections are not only restricted to occurrence in developed nations such as in US, Canada and Europe or Australia. However, there has been a lacuna in developing cost-effective meningococcal vaccines in those neglected areas of the World like African and Asian countries despite occurrence of varied meningococcal infections. Further, the individual vaccines available so far do not offer complete protection against all the serotypes in a specific region. There are still possibilities of varied Meningococcal infections within a given region, wherein the vaccines against particular serotype are not available at all. A particular subject vaccinated with a single or either two or three serotypes of the vaccine is still not completely protected against any potential infection due to a meningitidis serotype, which he or she has not been vaccinated with. Therefore, there is a necessity to develop more cost-effective meningitis vaccines which will offer prophylaxis against varied serotypes in a single combination vaccine against all meningitis serotypes irrespective of the nature of meningococcal infection. HAJJ pilgrims comprising the Muslim population from all over the World converge at Mecca every year. This increases the possibilities to contract varied meningitis infection by the Hajj pilgrims as the possibility of getting infected increases due to scarcity of hygienic food and water including sanitary considerations. It has been reported that at least 225 cases of meningococcal diseases are recorded each year after the Hajj season in Saudi Arabia due to the conglomeration of a large number of people from all over the world. Absence of potable drinking water also contributes to the risk of getting infected of typhoid fever due to infection by Salmonella typhi, in Hajj pilgrims. Therefore, apart from just preventing Meningitis, it is also essential to prevent the Hajj pilgrims from potential typhoid fever caused due to Salmonella typhi. The Centre for Disease Control and Prevention, a federal agency under the Department of Health and Human Services in United States has recommended vaccination for both Typhoid as well as Meningitis before travelling to the African meningitis belt. Further, the pilgrims going to Hajj or Umrah are compulsorily required to receive meningococcal vaccine, without which the visa for Hajj is not issued. Moreover, typhoid vaccines are recommended before making the journey. Therefore a combination vaccine that simultaneously satisfy this kind of a requirement, wherein travelers are immunized at the same time against meningitis and typhoid would reduce the necessity of taking two separate vaccines. Rather, the traveler or the pilgrim needs to get vaccinated by the combination vaccine so as to satisfy the requirements and increase vaccine compliance.
Meningococcal vaccines require bacterial fermentation of the polysaccharides using animal media and alcohol for downstream processing. Usage of animal sources of porcine origin and alcohol limits the applicability of Meningitis vaccine by several Hajj pilgrims, because there exists a possibility of retention of the animal components and alcohol in the final finished vaccine product. Intake of any product of porcine origin and using alcohol is forbidden under Islamic Shariat laws, considered as ‘HARAM’ in colloquial language. Hence, as a matter of personal choice, a majority of the Muslim population opts not to get vaccinated with the existing meningitis vaccines because of using porcine material in the fermentation media and further downstreaming through alcohol derived substances. Moreover, the product development of vaccine formulations against individual Meningococcal infections are expensive due to use of alcohols and animal resources used as raw materials in the bacterial fermentation and downstream processes. To overcome this situation, synthetic and semi synthetic medium free from animal source has been utilized for cultivation of Neisseria meningitidis in general.
Along with usage of such synthetic and semi-synthetic media, it has also been aimed to increase yield of the bacterial fermentation using various media sources in the prior state of the art. Different media compositions has been described in non-patent literature as mentioned in Frantz, J. Bact., 43:757-761.1942; and Catlin, J. Inf. Dis., 128(2):178-194, 1973: Watson Scherp Medium: J. Immunol 1958; 81:337-44. A modified media was used in Frantz and Catlin 6 Braz. J. Microbiol. 34: 2003. New Chemically Defined Medium (NCDM) is described by David bundal et al. JBC 1974 which resulted in Men X polysaccharide yield of 20 mg/L. There were several other prior arts available describing the media composition and cultivation strategies for increasing the capsule polysaccharide yield. The fermentation is carried out in batch and fed batch modes with different feeding strategy(ies) based on pH stat, spiking and constant rate as per the requirements of the cultivation of Meningitidis A, C, Y, W135, and X strains (henceforth represented as Men A, Men C, Men Y, Men W135, and Men X respectively). Some of these relevant prior arts are being discussed herein. Prior art U.S. Pat. No. 7,399,615 discloses omitting NH4Cl from fermentation media and replacing it with soy peptone (HSP-A; Nutricepts, inc; Minneapolis, Minn.). The fed batch 2 L fermentation batch with feed solution containing Heat shock protein HSP-A resulted in Men A polysaccharide yield of 1300-1400 mg/L at a maximum Optical Density (OD) between 14-20. Prior art U.S. Pat. No. 7,491,517 disclosed Neisseria meningitidis fastidious culture medium (NMFM) with CaCo3 to maintain pH 6.5 to 7.0 resulting in average yield of 30-40 mg/L with average OD of 10. Prior art WO2014/080423A2 disclosed the use of Casamino Acid instead of soy peptone or Yeast extract with novel feeding strategy using Feed Solution 1, (FS-1); and Feed Solution 2, (FS-2). To get Men X polysaccharides yield of 650 mg/L at 100 kDa stage when culture was harvested at a stage when culture OD is less than 60% of highest culture OD. The highest culture OD obtained was 16 at 580 nm.
The available fermentation medium and feed solutions described in prior arts utilized for cultivation and improving the yield of polysaccharide from serogroup A, C, Y, W135 and X had contributed only to some extent to increase the polysaccharide yield. But still there exists further requirement(s) of increasing the yield and decreasing the manufacturing cost of Neisseria meningitidis bacterial polysaccharides of all the strains viz. Men A, Men C, Men Y, Men W135, and Men X, for the purpose of making meningococcal vaccines further affordable and cost effective as well. The present invention is oriented to develop novel method(s) of increasing the bacterial polysaccharide of yield(s) of Men A, Men C, Men Y, Men W135, and Men X by using improved fermentation medium compositions containing peptones of non animal origin and utilizing novel feed components, and new methods of downstream processing and industrial purification of bacterial polysaccharides of Neisseria meningitidis. 
The vaccine formulations developed in this patent application specifically finds its application to the HAJJ pilgrims, who bear very high amount of risk of contracting infections from Meningitidis bacteria and typhoid fever, both of which are preventable through appropriate effective vaccination strategy comprising respective bacterial capsular polysaccharides as the vaccine antigens in a single combination vaccine including typhoid conjugate vaccine antigen. As already seen earlier, that none of the available meningitis vaccines cover all the existing serotypes of the disease. So, protection remains incomplete in almost every individual including even those who underwent vaccination against meningitis. Furthermore, a complete combination of all major existing serotypes of meningitis will comply with complete protection against meningitis for any individual hailing from any region of the World. Additionally, as there are no possibility(ies) of including any media components derived from animal origin, and alcohol, therefore, it is advantageous to term such a novel combination vaccine for Haj pilgrims, as a ‘HALAL’ vaccine preferably branded as HAJVAC™ by the patent applicant. Conjugate polysaccharide vaccines has been already developed specific to particular serotypes of Meningitidis infections, for long term immunity against the given serotypes. But, as said earlier conjugate vaccines does not ensure complete protection against all serotypes of the vaccine. Therefore, it is suggested that a polysaccharide based vaccine is adequately immunogenic to confer protection against any possible infection by any one or more of meningitidis or Salmonella typhi infections. The immunity conferred by polysaccharide vaccines last for a period of 2 years, and hence it is sufficient to justify the applicability of combined polysaccharide based vaccines as enumerated in this invention along with Vi polysaccharide conjugate typhoid vaccine antigen which are sufficient to ensure complete protection to the subjects in areas of high conglomeration of travelers in a specific geographic location.